Stroke is the third leading cause of death and the leading cause of long term disability in the US. Unfortunately, therapeutically targeting and preventing stroke-related damage from ischemic cascades and reperfusion injury has been challenging, making research on neuroprotective strategies a priority. Mammalian hibernation presents a unique opportunity to study naturally occurring neuroprotection. Hibernating ground squirrels undergo rapid and extreme changes in body temperature and brain perfusion as they cycle between lengthy torpor bouts (7-10 days) and brief periods of euthermia called interbout arousals (IBAs, <24 hours), but they do not suffer any residual brain damage. Arousal from torpor to IBA, where body temperature increases from 5 to 37oC and heart rate explodes from 3 to 300-400 beats/min, takes an average of just 2.8 hours. Different areas of the brain show markedly different activity profiles during hibernation, so neuroprotective differences must permit this spectrum of activity. This is of particular interest to stroke research, because neuroprotective strategies need to be focused specifically to the area of damage. Preliminary data from ground squirrel hypothalamic transcriptome sequencing shows that genes encoding regulatory proteins of well- known survival pathways, JAK3, STAT3, AKT1, MAPK1/3 are significantly upregulated during torpor and IBA. Additionally, production of the neuroprotective hormone melatonin accompanies arousal, suggesting that it plays a protective role at this time, when the squirrels are susceptible to reperfusion injury. Melatonin is known to be protective through survival pathways including JAK-STAT, AKT1, and MAPKs. In Specific Aim 1, I will administer the specific and competitive melatonin receptor antagonist luzindole to ground squirrels just prior to a natural arousal to determine melatonin's receptor-mediated protective effects. I will monitor the effects of luzindole on arousal parameters (body temperature and heart rate) using surgically implanted telemeters and measure levels of activated survival proteins in five differentially active areas of the brain upon arousal. A pilot study indicated that luzindole treatment delays arousal onset. In Specific Aim 2, I will establish the effect of the hibernator's environment on melatonin production. Normally, melatonin is only produced at night, inhibited by light during the day, but melatonin production upon arousal occurs regardless of the time of day. The dark and cold conditions during arousal from torpor and/or circadian clock action could be sufficient to trigger melatonin production. The hypothalamic transcriptome data indicates that expression of one circadian clock gene, DEC1, is elevated during torpor. To achieve this aim, I will artificially manipulate the photoperiod (light or dark) and temperature (4 or 25oC) of the hibernation environment and determine the effect on melatonin production and DEC1 expression. The results from these proposed experiments will provide new and valuable information on the potential of melatonin receptors as neuroprotective therapeutic targets, along with establishing a novel environmental and potential seasonal timing role for the clock gene DEC1. PUBLIC HEALTH RELEVANCE: Stroke victims currently have no neuroprotective treatment available to help prevent and combat ischemic and reperfusion damage that can occur even days after the stroke event. Hibernating ground squirrels provide a unique opportunity to examine a mammalian brain that is naturally neuroprotected, as it is subjected to periods of ischemia and reperfusion throughout hibernation. A better understanding of the protective molecular mechanisms underlying this natural tolerance could greatly benefit therapeutic approaches for stroke victims.